Life Sciences Commons^™

Tracking Insertion Mutants Within Libraries By Deep Sequencing And A Genome-Wide Screen For Haemophilus Genes Required In The Lung, Jeffrey D. Gawronski, Sandy M. S. Wong, Georgia Giannoukos, Doyle V. Ward, Brian J. Akerley

Brian J. Akerley

Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale "negative selection" technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model. Mutations in 136 genes resulted in defects in vivo, and quantitative estimates of fitness generated by this technique were in agreement with independent validation experiments using individual mutant strains. …